Unfolded protein-independent IRE1 activation contributes to multifaceted developmental processes in Arabidopsis

Abstract

AbstractAs an initial step for the unfolded protein response (UPR) pathway, the luminal domain of inositol requiring enzyme 1 (IRE1) senses unfolded proteins in the endoplasmic reticulum (ER). Recent findings in yeast and metazoans suggest alternative IRE1 activation without the sensor domain, although its mechanism and physiological significance remain to be elucidated. In Arabidopsis, the IRE1A and IRE1B double mutant (ire1a/b) is unable to activate cytoplasmic splicing of bZIP60 mRNA and regulated IRE1-dependent decay (RIDD) under ER stress, while the mutant does not exhibit severe developmental defects and is fertile under non-stress conditions. In this study, we focused on a third Arabidopsis IRE1 gene, designated as IRE1C, whose product lacks a sensor domain. We found that even though ire1c and ire1a/c mutants did not exhibit defective bZIP60 splicing and RIDD under ER stress, the ire1a/b/c triple mutant is lethal. Heterozygous IRE1C (ire1c/+) mutation in the ire1a/b mutants resulted in growth defects and reduction of the number of pollen grains. Genetic analysis revealed that IRE1C is required for male gametophyte development in the ire1a/b mutant background. Expression of a mutant form of IRE1B that lacks the luminal sensor domain (ΔLD) in the ire1a/b mutant did not complement defects in ER stress-dependent bZIP60 splicing and RIDD. Nevertheless, expression of ΔLD complemented a developmental defect in the male gametophyte in ire1a/b/c haplotype. In vivo, the ΔLD protein was activated by glycerol treatment that increases the composition of saturated lipid and was able to activate RIDD but not bZIP60 splicing. Phenotypes of IRE1B mutants lacking the sensor domain produced by CRISPR/Cas9-mediated gene editing in the ire1a/c mutant background were essentially same as those of ΔLD-expressing ire1a/b mutant. These observations suggest that IRE1 contributes to plant development, especially male gametogenesis, using an alternative activation mechanism that bypasses the unfolded protein-sensing luminal domain.